1. Field of the Invention
This invention relates to a process for the control of enteric bacterial pathogens in animals using prebiotic oligosaccharides.
2. Description of the Prior Art
Despite the efforts of researchers and public health agencies, the incidence of human infections from enteropathogenic bacteria such as Salmonella, enteropathogenic E. coli, and Campylobacter has increased over the past 20 years. For example, the number of actual reported cases of human Salmonella infection exceeds 40,000 per year. However, the Centers for Disease Control estimates that the true incidence of human Salmonella infections in the U.S. each year may be as high as 2 to 4 million. The USDA Economic Research Service has recently reported that the annual cost of the food borne illnesses caused by six common bacterial pathogens, Campylobacter spp., Clostridium perfringens, Escherichia coli 0157:H7, Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus, ranges from 2.9 billion to 6.7 billion dollars (Food Institute Report, USDA, AER, December, 1996). In addition to the impact of enteric pathogens on human health, many of these bacteria also cause significant infections in animals. For example, Salmonella infections in swine alone cost the United States swine industry more than 100 million dollars annually (Schwartz, 1990, “Salmonellosis in Midwestern Swine”, In: Proceedings of the United States Animal Health Assoc., pp. 443–449).
Animal food products remain a significant source of human infection by these pathogens. Contamination of meat and poultry with many bacterial food-borne pathogens, including the particularly onerous pathogens Campylobacter spp., Escherichia coli 0157:H7, and Salmonella spp., often occurs as a result of exposure of the animal carcass to ingesta and/or fecal material during or after slaughter. Any of the above-mentioned pathogens can then be transmitted to humans by consumption of meat and poultry contaminated in this manner.
Preharvest control of enteropathogenic bacteria is a high priority to the food industry. However, relatively few products have been developed to facilitate such efforts. One promising technique for preharvest pathogen control within the poultry industry has been the use of competitive exclusion cultures or probiotics (broadly defined as bacterial cultures which have a beneficial effect on the animal to which they are administered). Studies have typically focused on the evaluation of vaccines, establishment of protective normal intestinal flora, and the identification of feed additives that will inhibit the growth and colonization of enteropathogens such as Salmonella. 
It is well documented that the development of a normal intestinal microflora can increase resistance against Salmonella colonization of the gastrointestinal tract. In the poultry industry, oral inoculation of young chicks with probiotics which comprise anaerobic bacterial cultures of microflora prepared from the cecal contents or fecal droppings of mature chickens, has proven to effectively reduce Salmonella colonization (Snoeyenbos et al., Avian Dis., 1979, 23:904–913; Schneitz et al., Acta Pathol. Microbiol. Scand. Sect. B., 1981, 89:109–116; and Stavric et al., J. Food Prot., 1985, 48:778–782). These probiotics may decrease Salmonella colonization by rapidly colonizing the intestinal tract of the young chicks (Pivnick et al., J. Food Prot., 1981, 44:909–916), by competing for attachment sites on the intestinal wall (Snoeyenbos et al., ibid), or by producing bacteriostatic or bactericidal short-chained volatile fatty acids that inhibit the growth of enteropathogens (Barnes et al., J. Hyg. Camb., 1979, 82:263–283; Barnes et al., Am. J. Clin. Nutr., 1980, 33:2426–2433; Corrier et al., Avian Dis., 1990, 34:668–676; Corrier et al., Avian Dis., 1990, 34:617–625; and Hinton et al., Avian Dis., 1990, 34:626–633). Effective probiotics have been successfully developed for fowl and swine. For instance, Nisbet et al. of the USDA Agricultural Research Service, has developed a defined probiotic which is effective for controlling Salmonella colonization of swine (U.S. Pat. No. 5,951,977). Another probiotic has also developed by Nisbet et al. for the protection of fowl from Salmonella colonization (U.S. Pat. No. 5,478,557), and is sold commercially in the U.S. under the trademark PREEMPT (Milk Specialties Biosciences, Dundee, Ill.).
Immune lymphokines (ILK) have also been developed for protecting poultry from colonization with enteric pathogens as described by Ziprin et al. (1989, Poult. Sci., 68:1637–1642), McGruder et al. (1993, Poult. Sci., 72:2264–2271), Ziprin et al. (1996, Avian Dis., 40:186–192), and Tellez et al. (1993, Avian Dis., 37:1062–1070), and more recently by Kogut et al. (U.S. Pat. Nos. 5,891,443 and 5,691,200). Most recently, Anderson et al. (U.S. Pat. No. 6,475,527) disclosed that chlorates substantially reduce populations of enteropathogenic bacteria in the alimentary tract when administered orally, or alternatively, reduce the populations of these enteropathogens present as contaminants on the surface of the animals following external application of chlorates.
Several oligosaccharides have been described as having prebiotic activity in foods, animal feeds, and cosmetics. Like the above-mentioned probiotics, prebiotics also control enteropathogens by facilitating the establishment of protective normal intestinal flora. However, in contrast with the probiotics which consist of viable microorganisms, the prebiotics are oligosaccharides which assist the establishment of populations of the normal protective flora but not enteropathogenic bacteria by providing a substrate which may be readily utilized by beneficial bacteria but not by the enteropathogens. Glucooligosaccharides exhibit certain desirable characteristics in these applications, particularly in their ability to support the growth of beneficial probiotic normal flora bacteria without the generation of undesirable amounts of gases (Valette et al., Sci. Food Agric., 1993, 62:121–127). Besides maltooligosaccharides, other glucooligosaccharides include isomaltooligosaccharides, kojioligosaccharides, and mixtures of variously linked saccharides (Yatake, In Oligosaccharides. Production, Properties and Applications; Nakakuki Ed.; Japanese Technology Reviews, Section E: Biotechnology, Vol. 3, No. 2; Gordon and Breach Science Publishers, Yverdon, Switzerland, 1993, pp. 79–89).
One group of glucooligosaccharides that is garnering an increasing amount of interest includes those synthesized from sucrose via glucansucrases (Castillo et al., Ann. NY Acad. Sci., 1992, 672:425–430). Glucansucrases are typically extracellular enzymes secreted by bacteria such as Streptococcus, Lactobacillus, and Leuconostoc spp. They act by transferring D-glucosyl units from sucrose to D-glucose polymers, with the concomitant release of D-fructose. Glucansucrases, including dextransucrase and alternansucrase, have been reviewed in detail (Robyt, Adv. Carbohydr. Chem. Biochem., 1995, 51:133–168; Robyt, In Enzymes for Carbohydrate Engineering, Park, Robyt, and Choi Eds, Elsevier Sciences, Amsterdam, 1996, pp. 1–22; Monchois et al., FEMS Microbiol. Rev., 1999, 23:131–151; Remaud-Simeon et al., J. Molec. Catalysis B: Enzymatic, 2000, 10:117–128; and Côté, In Biopolymers, Vol. 5: Polysaccharides I: Polysaccharides from Prokaryotes, Vandamme, DeBaets, and Steinbüchel Eds., Wiley-VCH, Weinheim, Germany, 2002, pp. 323–350).
of particular interest are the so-called acceptor reactions of glucansucrases. In an acceptor reaction, D-glucosyl units are transferred from sucrose to a hydroxyl-bearing acceptor molecule, resulting in the formation of an α-D-glucopyranosyl acceptor product. Most known acceptors are carbohydrates, although non-carbohydrate acceptors have been described. Alternansucrase, in particular, is known for its ability to catalyze a wide variety of acceptor reactions with various sugars. Unlike dextransucrase (Robyt and Eklund, Carbohydr. Res., 1983, 121:279–286; and Yamauchi and Ohwada, Agric. Biol. Chem., 1969, 33:1295–1300), alternansucrase often forms two or more linkage types with a single acceptor (Castillo et al., ibid; Côté and Robyt, Carbohydr. Res., 1982, 111:127–142; and Pelenc et al., Sciences des Aliments, 1991, 11:465–476). It has also been noted that alternansucrase carries out acceptor reactions with a wider variety of acceptors with greater yields (Argüello-Morales et al., Carbohydr. Res., 2001, 331:403–411).
However, despite these and other advances, the need persists for technologies for controlling enteric pathogens in animals.